Oligosaccharide composition for treating skin diseases

ABSTRACT

The invention discloses a composition comprising at least one N-acetyl-lactosamine, at least one sialylated oligosaccharide and at least one fucosylated oligosaccharide, for use in the prevention and/or treatment of skin conditions and skin diseases. Preferably said composition is a starter infant formula. Said skin disease is in particular atopic dermatitis.

This invention relates to a composition for use in the prevention and/ortreatment of skin conditions, such as the promotion of skin health, andskin diseases, particularly atopic dermatitis.

BACKGROUND OF THE INVENTION

Atopic dermatitis is a chronic itchy skin disease that is common inchildren but may occur at any age. It is also known as eczema or atopiceczema. There is a strong association between food allergy and atopicdermatitis in the age group of young children, and food allergies areoften suspected in children with atopic dermatitis.

Atopic dermatitis usually occurs in people who have an atopic tendency.This means they may develop any or all of three closely linkedconditions: atopic dermatitis, asthma and hay fever (allergic rhinitis).

A phenomenon of atopic dermatitis occurs as follows. Patches ofsensitive skin flare up in a rash in response to certain triggers. Thesetriggers vary from person to person. In the case of infants and youngchildren, the list of common triggers to watch for includes cow's milkand other possible ingredients of infant formula such as wheat or soy.Atopic dermatitis can become a vicious cycle. Something irritates thechild skin, making it red and inflamed. It itches, the child scratchesit, and the skin becomes more inflamed. The outer protective layer ofthe skin is lost, and the affected area becomes even more sensitive toirritants and dries out easily. The infant continues to be exposed towhatever it was that triggered these episodes in the first place. Therash develops further and the cycle perpetuates itself.

There is no known single cause for atopic dermatitis. It probablyreflects more than one condition. There are many theories regarding theunderlying mechanisms. Current research is investigating the role offilaggrin gene mutations, defects in skin cells (keratinocytes), theimmune system, skin surface microbes (bacteria, viruses and yeasts), andmany other factors.

All skin conditions and skin diseases can affect the general populationor the population of persons at risk of allergies or the population ofallergic (hence sick) persons.

Such skin conditions and skin diseases, and in particular atopicdermatitis, are of particular importance for infants, babies or childrenas they have a sensitive skin that undergoes an intense growth andphases of multiplication, rendering it even more susceptible to skindiseases. The population of infants without history of allergies intheir family, and who become allergic, is increasing.

Hypoallergenic foodstuff is a type of food which is unlikely to causeallergic reactions. Hypoallergenic foodstuffs have been developed, inparticular for infant formulae, because infants and children arebecoming increasingly likely to develop allergy in the firstmonths/years of their life.

Hypoallergenic infant formulae are used to help, prevent and treatallergic diseases and particularly food allergies in infants andoccasionally in young children. They are generally made without the useof common allergens like wheat, soy and dairy products. They alsousually derive from cow's milk, however because of the way theirproteins have been broken down, they are well tolerated by most infants.

Hypoallergenic infant formulae generally come in three main varieties:partially hydrolysed, extensively hydrolysed and free amino-acid-based.Hydrolyzed infant formulae have the larger protein chains broken downinto shorter, easy to digest proteins, whereas free amino-acid formulaedo not include protein chains at all, but rather contain all the basicamino acids. Partially hydrolysed infant formulae differ fromextensively hydrolysed infant formulae in that their protein chains canbe longer.

Hypoallergenic infant formulae have the drawback that their cost is muchhigher than the cost of regular cow's milk formulae.

However, there is still a need for hypoallergenic infant formulae forthe prevention and/or treatment of skin conditions and skin diseasesthat infant and even young children can develop, among which atopicdermatitis.

Human milk oligosaccharides (HMOs) are, collectively, the third largestsolid constituents in human milk, after lactose and fat. HMO usuallyconsists of lactose at the reducing end with a carbohydrate core thatoften contains a fucose or a sialic acid at the non-reducing end. Thereare approximately one hundred milk oligosaccharides that have beenisolated and characterized, however these represent only a very smallportion of the total number remaining to be characterized.

In the past, infant formulae were developed using HMO ingredients, suchas fucosylated oligosaccharides, lacto-N-tetraose, lacto-N-neotetraose,or sialylated oligosaccharides, for different purposes.

EP 0 975 235 B1 from Abbott Laboratories describes a syntheticnutritional composition comprising one or more human milkoligosaccharides, wherein the HMOs in the composition are chosen among agroup of eight HMOs (3-fucosyllactose, lacto-N-fucopentaose III,lacto-N-fucopentaose II, difucosyllactose, 2′-fucosyllactose,lacto-N-fucopentaose I, lacto-N-neotetraose and lacto-N-fucopentaose V)wherein said composition is intended for cases of normal, healthyinfants, children, adults or individuals having specialized needs suchas those that accompany certain pathological conditions. This Europeanpatent states that, generally speaking, oligosaccharides protect infantsfrom viral and bacterial infections of the respiratory, gastrointestinaland uro-genital tracts. Nothing is mentioned concerning the issue ofatopic dermatitis and more generally the prevention and/or treatment ofskin disease.

From the foregoing, it may be seen that there is a need for an effectivenutritional composition for the prevention of secondary atopicdermatitis or the promotion of skin health, particularly in infants andyoung children, and which may be conveniently and safely administered.

There is a need for an improvement of skin conditions or skin diseases,such as atopic dermatitis, by a non-drug-based intervention that iscompatible with fragile individuals like infants or babies.

There is a need for a long term effect in the reduction of thefrequency, occurrence, severity and/or duration of such skin conditionsand skin diseases. There is furthermore a need for an effect thatbecomes measurable “later in life”, especially some years after theintervention.

There is a need for a food intervention to infants, babies and children,targeted at risks of allergy or not, that induces a reduction ofallergic manifestations, especially on the skin.

There is a need for such intervention that induces the maintenance orthe improvement of skin health.

SUMMARY OF THE INVENTION

The present inventors have found surprisingly that the administration ofa mixture of specific human oligosaccharides is particularly effectivein the prevention and/or treatment of skin conditions and skin diseases,and in particular in the prevention and/or treatment of atopicdermatitis, and/or in the promotion of skin health.

Accordingly, the present invention provides a composition, comprising atleast one N-acetyl lactosamine, at least one sialylated oligosaccharideand at least one fucosylated oligosaccharide, for use in the preventionand/or treatment of skin conditions and skin diseases, preferably atopicdermatitis.

The composition is preferably a synthetic nutritional composition. Thecomposition comprises three different types of uses as syntheticnutritional composition. In the first case, individuals and particularlyinfants are healthy, without any risk of allergy because of no historyof allergies in their family. In the second case, individuals andparticularly infants are healthy, but at risk of allergy because ofhistory of allergies in their family. In the third case, individuals andparticularly infants are allergic, and hence sick. The first case is thepreferred target according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms have the following meanings.

The term “infant” means a child under the age of 12 months.

The term “young child” means a child aged between one and three years.

The term “infant formula” means a foodstuff intended for particularnutritional use by infants during the first four to six months of lifeand satisfying by itself the nutritional requirements of this categoryof person (Article 1.2 of the European Commission Directive 91/321/EECof May 14, 1991 on infant formulae and follow-on formulae).

The term “follow-on formula” means a foodstuff intended for particularnutritional use by infants aged over four months and constituting theprincipal liquid element in the progressively diversified diet of thiscategory of person.

The term “starter infant formula” means a foodstuff intended forparticular nutritional use by infants during the first four months oflife.

The term “baby food” means a foodstuff intended for particularnutritional use by infants during the first years of life.

The term “infant cereal composition” means a foodstuff intended forparticular nutritional use by infants during the first years of life.

The term “growing-up milk” means a milk-based beverage adapted for thespecific nutritional needs of young children.

The term “weaning period” means the period during which the mother'smilk is substituted by other food in the diet of an infant.

The term “skin disease” means atopic dermatitis and other related skinissues. Eczema is an atopic dermatitis.

The term “prevention and/or treatment of skin diseases” means theprevention and the reduction of frequency and/or occurrence and/orseverity and/or duration of skin diseases, i.e. atopic dermatitis andother related skin issues, in particular atopic dermatitis. Occurrenceis related to the number of any skin disease. Frequency is related tothe number of the same skin disease. This prevention encompasses thereduction of frequency and/or of severity of said skin diseases later inlife. The term “later in life” encompasses the effect after thetermination of the intervention. The effect “later in life” can bepreferably 2 to 4 weeks, 2 to 12 months or years (e.g. 2, 5, 10 years)after the termination of said intervention.

The term “skin conditions” means conditions that irritate, clog orinflame the skin. Skin conditions can cause symptoms of skin diseasessuch as redness, swelling, burning and itching.

The term “prevention and/or treatment of skin conditions” means thepromotion of skin health and/or the prevention of skin dehydrationand/or the enhancement of the hydration of the skin and/or the reductionof skin rash, roughness and/or dryness. This prevention furtherencompasses the establishment of a phenotype, visible or hidden, thataccompany the reduction of frequency, occurrence, severity and/orduration of said skin conditions later in life.

The term “enhancement of the oral tolerance to allergens” means thereduction of the sensibility to allergens when taken orally.

The term “nutritional composition” means a composition which nourishes asubject. This nutritional composition is usually to be taken orally orintraveinously, and it usually includes a lipid or fat source and aprotein source.

The term “synthetic mixture” means a mixture obtained by chemical and/orbiological means, which can be chemically identical to the mixturenaturally occurring in mammalian milks. A composition is said to besynthetic as soon as at least one of its components is obtained bychemical and/or biological (e.g. enzymatic) means.

The term “hypoallergenic nutritional composition” means a nutritionalcomposition which is unlikely to cause allergic reactions.

The term “sialylated oligosaccharide” means an oligosaccharide having asialic acid residue.

The term “fucosylated oligosaccharide” means an oligosaccharide having afucose residue.

The term “prebiotic” means non-digestible carbohydrates thatbeneficially affect the host by selectively stimulating the growthand/or the activity of healthy bacteria such as bifidobacteria in thecolon of humans (Gibson G R, Roberfroid M B. Dietary modulation of thehuman colonic microbiota: introducing the concept of prebiotics. J.Nutr. 1995; 125:1401-12).

The term “probiotic” means microbial cell preparations or components ofmicrobial cells with a beneficial effect on the health or well-being ofthe host. (Salminen S, Ouwehand A. Benno Y. et al. “Probiotics: howshould they be defined” Trends Food Sci. Technol. 1999:10 107-10).

An “allergy” is an allergy which has been detected by a medical doctorand which can be treated occasionally or in a more durable manner.

All percentages are by weight unless otherwise stated.

The composition according to the invention is preferably ahypoallergenic composition.

Said composition contains at least one N-acetyl-lactosamine. That is tosay that the composition according to the invention containsN-acetyl-lactosamine and/or an oligosaccharide containingN-acetyl-lactosamine. Suitable oligosaccharides containingN-acetyl-lactosamine include lacto-N-tetraose (LNT) andlacto-N-neotetraose (LNnT).

Thus, according to the invention, the N-acetyl-lactosamine is preferablyselected from the group comprising lacto-N-tetraose (LNT) andlacto-N-neotetraose (LNnT).

LNT and LNnT may be synthesised chemically by enzymatic transfer ofsaccharide units from donor moieties to acceptor moieties usingglycosyltransferases as described for example in U.S. Pat. No. 5,288,637and WO 96/10086. Alternatively, LNT and LNnT may be prepared by chemicalconversion of Keto-hexoses (e.g. fructose) either free or bound to anoligosaccharide (e.g. lactulose) into N-acetylhexosamine or anN-acetylhexosamine-containing oligosaccharide as described in Wrodnigg,T. M.; Stutz, A. E. (1999) Angew. Chem. Int. Ed. 38:827-828.N-acetyl-lactosamine produced in this way may then be transferred tolactose as the acceptor moiety.

Preferably the composition according to the invention contains from 0.1to 3 g N-acetyl-lactosamine per 100 g of composition on a dry weightbasis.

According to the invention, the sialylated oligosaccharide is selectedfrom the group comprising 3′-sialyllactose and 6′-sialyllactose.Preferably, both 3′-sialyllactose and 6′-sialyllactose are present insaid composition. In this embodiment, the ratio between 3′-sialyllactoseand 6′-sialyllactose lies preferably in the range between 5:1 and 1:2.

The 3′- and 6′-forms of sialyllactose may be isolated by chromatographicor filtration technology from a natural source such as animal milks.Alternatively, they may be produced by biotechnological means usingspecific sialyltransferases or sialidases, neuraminidases, either by anenzyme based fermentation technology (recombinant or natural enzymes),by chemical synthesis or by a microbial fermentation technology. In thelatter case microbes may either express their natural enzymes andsubstrates or may be engineered to produce respective substrates andenzymes. Single microbial cultures or mixed cultures may be used.Sialyl-oligosaccharide formation can be initiated by acceptor substratesstarting from any degree of polymerisation (DP), from DP=1 onwards.Alternatively, sialyllactoses may be produced by chemical synthesis fromlactose and free N′-acetylneuraminic acid (sialic acid). Sialyllactosesare also commercially available for example from Kyowa Hakko Kogyo ofJapan.

Preferably the composition according to the invention contains from 0.05to 2 g, more preferably 0.1 to 2 g, of sialylated oligosaccharide(s) per100 g of composition on a dry weight basis.

The fucosylated oligosaccharide may be selected from the groupcomprising 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose,lacto-N-fucopentaoses (that is to say lacto-N-fucopentaose I,lacto-N-fucopentaose II, lacto-N-fucopentaose III andlacto-N-fucopentaose V), lacto-N-difucohexaose I,fucosyllacto-N-hexaose, Difucosyllacto-N-hexaose I andDifucosyllacto-N-neohexaose II. A particularly preferred fucosylatedoligosaccharide is 2′-fucosyllactose (2-FL).

The fucosylated oligosaccharide may be isolated by chromatography orfiltration technology from a natural source such as animal milks.Alternatively, it may be produced by biotechnological means usingspecific fucosyltransferases and/or fucosidase either through the use ofenzyme-based fermentation technology (recombinant or natural enzymes) ormicrobial fermentation technology. In the latter case, microbes mayeither express their natural enzymes and substrates or may be engineeredto produce respective substrates and enzymes. Single microbial culturesand/or mixed cultures may be used. Fucosylated oligosaccharide formationcan be initiated by acceptor substrates starting from any degree ofpolymerization (DP), from DP=1 onwards. Alternatively, fucosylatedoligosaccharides may be produced by chemical synthesis from lactose andfree fucose. Fucosylated oligosaccharides are also available for examplefrom Kyowa Hakko Kogyo of Japan.

Preferably, the composition according to the invention contains from 0.1to 3 g of fucosylated oligosaccharide(s) per 100 g of composition on adry weight basis.

In a preferred embodiment, the composition of the invention comprisesfrom 0.05 to 3 g of the total amount of N-acetylated lactosamine(s),sialylated oligosaccharide(s) and fucosylated oligosaccharide(s) per 100g of composition.

The composition of the invention can further comprise at least oneprobiotic bacterial strain, said probiotic bacterial strain preferablybeing Bifidobacteria and/or Lactobacilli.

Suitable probiotic bacterial strains include Lactobacillus rhamnosusATCC 53103 available from Valio Oy of Finland under the trademark LGG,Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCMI-2116, Lactobacillus reuteri sold by BioGaia A.B under the trademarkReuteri, Lactobacillus johnsonii CNCM I-1225, Streptococcus salivariusDSM 13084 sold by BLIS Technologies Limited of New Zealand under thedesignation Kl2, Bifidobacterium lactis CNCM 1-3446 sold inter alia bythe Christian Hansen company of Denmark under the trademark Bb 12,Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co.Ltd. of Japan under the trademark BB536, Bifidobacterium breve sold byDanisco under the trademark Bb-03, Bifidobacterium breve sold byMorinaga under the trade mark M-16V, Bifidobacterium infantis sold byProcter & Gamble Co. under the trademark Bifantis and Bifidobacteriumbreve sold by Institut Rosell (Lallemand) under the trademark R0070.

Preferably, the composition according to the invention contains from10e3 to 10e12 cfu of probiotic bacterial strain, more preferably between10e7 and 10e12 cfu, per g of composition on a dry weight basis.

The composition of the invention can further comprise at least oneprebiotic, usually in an amount between 0.3 and 10% by weight ofcomposition.

Prebiotics are usually non-digestible in the sense that they are notbroken down and absorbed in the stomach or small intestine and thusremain intact when they pass into the colon where they are selectivelyfermented by the beneficial bacteria. Examples of prebiotics includecertain oligosaccharides, such a fructooligosaccharides (FOS) andgalactooligosaccharides (GOS). A combination of prebiotics may be usedsuch as 90% GOS with 10% short chain fructo-oligosaccharides such as inthe product by BENEO-Orafti sold under the trademark “Orafti®oligofructose” (seehttp://www.beneo-orafti.com/Our-Products/Oligofructose) (previouslyRaftilose®) or 10% inulin such as in the product sold by BENEO-Oraftiunder the trademark “Orafti® inulin” (seehttp://www.beneo-orafti.com/Our-Products/Inulin) (previouslyRaftiline®). A particularly preferred combination of prebiotics is 70%short chain fructo-oligosaccharides and 30% inulin, which is a productsold by BENEO-Orafti under the trademark “Prebio 1”.

The composition according to the invention can further comprise othernutritional compounds that synergize with the claimed oligosaccharidesto deliver the claimed benefits on skin, such as skin allergies oratopic dermatitis. Such additional compounds can be for example lowallergenic proteins (i.e. hydrolyzed or partly hydrolyzed proteins), lowlactose ingredients or low lactose saccharides. It is postulated thatthe synergy of the ingredients may be established by the fact that theingredients all together help to maintain a low physiological level ofan effector that stays below therehold where skin effects (such asatopic dermatitis) starts to show.

The composition according to the invention is preferably a syntheticnutritional composition. In this case, it can be a starter infantformula, an infant formula, a baby food, an infant cereal composition, afollow-on formula or a growing-up milk, and said composition ispreferably a starter infant formula.

According to a preferred embodiment, the composition according to theinvention is for use in healthy infants and healthy young children.

The composition according to the invention can be for use before and/orduring a weaning period.

Thus preferably the composition according to the invention is for use inthe reduction of frequency and/or occurrence and/or severity and/orduration of atopic dermatitis and/or for use in the promotion of skinhealth.

In an implementation, the composition according to the invention is foruse in the promotion of skin health and/or for the prevention of skindehydration and/or for use in the enhancement of the hydration of theskin and/or for use in the reduction of skin rash, roughness and drynessand/or for use in the enhancement of the oral tolerance to allergens.

The invention includes also the use of a composition comprising at leastone N-acetyl lactosamine, at least one sialylated oligosaccharide and atleast one fucosylated oligosaccharide, as a synthetic nutritional agent,for the prevention and/or treatment of skin conditions and skindiseases, preferably atopic dermatitis.

The invention includes also the use of a composition comprising at leastone N-acetyl lactosamine, at least one sialylated oligosaccharide and atleast one fucosylated oligosaccharide, as a synthetic nutritional agent,for enhancement of the oral tolerance to allergens.

These uses encompass the case where the composition is a supplement,preferably provided in the form of unit doses.

All the uses stated above are particularly intended for infants andyoung children. The compositions and uses as per the present inventionare particularly suited for infants and children at risk of allergies,having a family history of allergies, or having already experienced someepisodes of allergies (especially respiratory allergies or skinallergies). In one embodiment the composition and uses of the inventionapply to teenagers or adults at risk of allergies or having experiencesepisodes of allergies (especially respiratory allergies or skinallergies).

Without wishing to be bound by theory, the inventors believe that theefficacy of the combination of oligosaccharides described above in theprevention and/or treatment of skin conditions and skin diseases,particularly atopic dermatitis, may be the result of the synergisticcombination of mucosal immunity modulator effects triggered by lacticacid bacteria through their stimulation with a specific oligosaccharideblend. The inventors believe the establishment of mucosa associatedmicrobiota at large with minimal inflammatory stimulation is achieved bythe selective promotion of beneficial commensal lactic acid bacteriawith simultaneous modulation of inflammatory regulators such asgalectins (e.g. Galectin-1 and galectin-3). Moreover, such compositionmay modulate the metabolism of endogenous microbiota leading toproduction of short-chain fatty acid that will contribute to activateimmune cells underlying the gut mucosa. Following triggering of themucosal immune system, activated immune cells, immune-active compoundsand/or immune mediators will circulate to distal locations, includingskin, where they will exert immune modulatory activity. All together,these mechanisms will contribute to balance potential skin inflammatoryconditions leading to improvement of related clinical manifestationssuch as dermatitis and eczema. Further, the inventors believe that suchorchestrated immunity translates in lower skin sensitivity towardsexogenous irritants and better skin barrier function leading to lowerskin rash, roughness, and dryness, associated with reactive skin.

The oligosaccharides may be administered in the same composition or maybe administered sequentially.

If the age group of 0 to 12 months of life is to be addressed, thecomposition is preferably a nutritional composition consumed in liquidform. It may be a nutritionally complete formula such as an infantformula, a follow-on formula or a growing-up milk.

Alternatively for the group of young children group, the composition maybe a juice drink or other chilled or shelf stable beverage or a soup,for example, or a baby food, or an infant cereal composition.

The composition according to the invention also contains a proteinsource, preferably in an amount below 2.0 g per 100 kcal, even morepreferably in an amount below 1.8 g per 100 kcal. The type of protein isnot believed to be critical to the present invention provided that theminimum requirements for essential amino acid content are met andsatisfactory growth is ensured. Thus, protein sources based on whey,casein and mixtures thereof may be used as well as protein sources basedon soy. As far as whey proteins are concerned, the protein source may bebased on acid whey or sweet whey or mixtures thereof and may includealpha-lactalbumin and beta-lactoglobulin in any desired proportions.

The composition according to the present invention generally contains acarbohydrate source. This is particularly preferable in the case wherethe nutritional composition of the invention is an infant formula. Inthis case, any carbohydrate source conventionally found in infantformulae such as lactose, saccharose, maltodextrin, starch and mixturesthereof may be used although the preferred source of carbohydrates islactose.

The composition according to the present invention generally contains asource of lipids. This is particularly relevant if the nutritionalcomposition of the invention is an infant formula. In this case, thelipid source may be any lipid or fat which is suitable for use in infantformulae. Preferred fat sources include palm oleic, high oleic sunfloweroil and high oleic safflower oil. The essential fatty acids linoleic anda-linolenic acid may also be added as may small amounts of oilscontaining high quantities of preformed arachidonic acid anddocosahexaenoic acid such as fish oils or microbial oils. The fat sourcepreferably has a ratio of n-6 to n-3 fatty acids of about 5:1 to about15:1; for example about 8:1 to about 10:1.

The composition of the invention also contains preferably all vitaminsand minerals understood to be essential in the daily diet and innutritionally significant amounts. Minimum requirements have beenestablished for certain vitamins and minerals. Examples of minerals,vitamins and other nutrients optionally present in the composition ofthe invention include vitamin A, vitamin B1, vitamin B2, vitamin B6,vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid,inositol, niacin, biotin, pantothenic acid, choline, calcium,phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine,potassium, sodium, selenium, chromium, molybdenum, taurine, andL-carnitine. Minerals are usually added in salt form. The presence andamounts of specific minerals and other vitamins will vary depending onthe intended population.

If necessary, the composition of the invention may contain emulsifiersand stabilisers such as soy, lecithin, citric acid esters of mono- anddi-glycerides, and the like.

The composition of the invention may also contain other substances whichmay have a beneficial effect such as lactoferrin, nucleotides,nucleosides, and the like.

A composition according to the invention will now be described by way ofexample.

The formula may be prepared in any suitable manner. For example, it maybe prepared by blending together the protein, the carbohydrate source,and the fat source in appropriate proportions. If used, the emulsifiersmay be included at this point. The vitamins and minerals may be added atthis point but are usually added later to avoid thermal degradation. Anylipophilic vitamins, emulsifiers and the like may be dissolved into thefat source prior to blending. Water, preferably water which has beensubjected to reverse osmosis, may then be mixed in to form a liquidmixture. The temperature of the water is conveniently in the rangebetween about 50° C. and about 80° C. to aid dispersal of theingredients. Commercially available liquefiers may be used to form theliquid mixture. The N-acetyl-lactosamine(s), sialylatedoligosaccharide(s) and fucosylated oligosaccharide(s) will be added atthis stage if the final product is to have a liquid form. If the finalproduct is to be a powder, the oligosaccharides may likewise be added atthis stage if desired. The liquid mixture is then homogenised, forexample in two stages.

The liquid mixture may then be thermally treated to reduce bacterialloads, by rapidly heating the liquid mixture to a temperature in therange between about 80° C. and about 150° C. for a duration betweenabout 5 seconds and about 5 minutes, for example. This may be carriedout by means of steam injection, an autoclave or a heat exchanger, forexample a plate heat exchanger.

Then, the liquid mixture may be cooled to between about 60° C. and about85° C. for example by flash cooling. The liquid mixture may then beagain homogenised, for example in two stages between about 10 MPa andabout 30 MPa in the first stage and between about 2 MPa and about 10 MPain the second stage. The homogenised mixture may then be further cooledto add any heat sensitive components, such as vitamins and minerals. ThepH and solids content of the homogenised mixture are convenientlyadjusted at this point.

The homogenised mixture is transferred to a suitable drying apparatussuch as a spray dryer or freeze dryer and converted to powder. Thepowder should have a moisture content of less than about 5% by weight.The N-acetyl-lactosamine(s), sialylated oligosaccharide(s) andfucosylated oligosaccharide(s) may be added at this stage by dry-mixingalong with the probiotic bacterial strain(s) if used, or by blendingthem in a syrup form of crystals, along with the probiotic bacterialstrain(s) if used, and spray-dry (or freeze-dry).

If a liquid composition is preferred, the homogenised mixture may besterilised then aseptically filled into suitable containers or may befirst filled into the containers and then retorted.

In another embodiment, the composition of the invention may be asupplement including the N-acetyl-lactosamine(s), sialylatedoligosaccharide(s) and fucosylated oligosaccharide(s) in an amountsufficient to achieve the desired effect in an individual. This form ofadministration is more suited to older children and adults. Preferablythe daily dose of the N-acetyl-lactosamine(s) is from 0.1 to 3 g, thedaily dose of the sialylated oligosaccharide(s) is from 0.1 to 2 g, andthe daily dose of the fucosylated oligosaccharide(s) is from 0.1 to 3 g.

The amount of oligosaccharides to be included in the supplement will beselected according to the manner in which the supplement is to beadministered. For example, if the supplement is to be administered twicea day, each supplement may contain 0.05 to 1.5 g ofN-acetyl-lactosamine, 0.05 to 1 g of sialylated oligosaccharide and 0.05to 1.5 g of fucosylated oligosaccharide.

The supplement may be in the form of tablets, capsules, pastilles or aliquid for example. The supplement may further contain protectivehydrocolloids (such as gums, proteins, modified starches), binders, filmforming agents, encapsulating agents/materials, wall/shell materials,matrix compounds, coatings, emulsifiers, surface active agents,solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents,carriers, fillers, co-compounds, dispersing agents, wetting agents,processing aids (solvents), flowing agents, taste masking agents,weighting agents, jellifying agents and gel forming agents. Thesupplement may also contain conventional pharmaceutical additives andadjuvants, excipients and diluents, including, but not limited to,water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc,sugars, starch, gum arabic, vegetable oils, polyalkylene glycols,flavouring agents, preservatives, stabilizers, emulsifying agents,buffers, lubricants, colorants, wetting agents, fillers, and the like.

Further, the supplement may contain an organic or inorganic carriermaterial suitable for oral or parenteral administration as well asvitamins, minerals trace elements and other micronutrients in accordancewith the recommendations of Government bodies such as the USRDA.

An example of the composition of an infant formula according to thepresent invention is given below. This composition is given by way ofillustration only. Another example is based on commercial NAN and/orlactogen Infant formulae (from Nestlé, Switzerland) to which thespecific oligosaccharides of the invention are added as in the amountstated below.

Nutrient per 100 kcal per litre Energy (kcal) 100 670 Protein (g) 1.8312.3 Fat (g) 5.3 35.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg)101 675 Lactose (g) 11.2 74.7 Prebiotic (70% FOS, 30% 0.64 4.3 insulin)(g) Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg) 64 430 Ca(mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (μg) 8 50 Se (μg) 2 13 VitaminA (μg RE) 105 700 Vitamin D (μg) 1.5 10 Vitamin E (mg TE) 0.8 5.4Vitamin K1 (μg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50Folic acid (μg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (μg) 0.3 2Biotin (μg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (μg) 15 100 Cu(mg) 0.06 0.4 Zn (mg) 0.75 5 3′sialyllactose (mg) 30 200 6′sialyllactose(mg) 6 40 LNnT (mg) 30 200 2FL (g) 0.3 2.0

Experimental Data: Finding Summary

The findings outlined below show that a specific blend ofoligosaccharides promotes the metabolic acitivity and growth of lacticacid bacteria, such as Bifidobacterium longum subsp infantis. Withoutwishing to be bound by theory, this effect might in part explain theobservation that relatively low amounts of such specific oligosaccharideblends were found in early milk of mothers, whose cesarean born childrendeveloped atopic allergies and especially atopic eczema up to the age of2 years. This indicates that, according to the invention, providing anutritional composition with a secured level of the specificoligosaccharides blend may help reestablishing the natural balance ofbacteria in the intestinal tract of infants and/or thus positivelyinfluence the health status in regards to the prevention and/ortreatment of skin conditions and skin diseases, preferably atopicdermatitis

Study Model 1: Stimulation of Bifidobacterium longum Subsp Infantis

Approach:

Bifidobacterium longum subsp infantis (ATCC15697) was grown anaerobic inAPI growth medium supplemented either with 1% (w/v) glucose, or 1% (w/v)2′fucosyllactose (2FL) or 1% (w/v) lacto-N-neotetrasose (LNnT) or 1%(w/v) 6′sialyllactose (6SL) or 1% (w/v) of a combination of equalamounts of 2FL, LNnT and 6SL. Each overnight culture was diluted to havea starting OD600 of 0.1 in DMEM (Dulbeccos modified Eagle Medium)containing as a carbon source 0.1% glucose. This medium was used withoutany further carbohydrate supplement or with additional 1% (w/v) glucose,or 1% (w/v) 2′fucosyllactose (2FL) or 1% (w/v) lacto-N-neotetrasose(LNnT) or 1% (w/v) 6′sialyllactose (6SL) or 1% (w/v) of a combination ofequal amounts of 2FL, LNnT and 6SL. Conditioning of DMEM media was thusdone at 37° C. anaerobic.

After another over night incubation, growth of bacteria was monitored bymeasuring OD at 600 nm. Conditioned media were then centrifuged andsupernatants filtered through a 0.22 micrometer filter to removebacteria. Acetate in conditioned media was quantified by HPLC using aHi-Plex H column and a UV detector.

Results:

We surprisingly found that an oligosaccharide blend composed of equalparts of a fucoslylated oligosaccharide (e.g. 2′FL), an N-acetylatedoligosaccharide (e.g. LNnT) and a sialylated oligosaccharide (e.g. 6SL)significantly increased metabolic activity of a lactic acid bacterium(e.g. bifidobacterium), as seen by the formation of acetate (FIG. 1).

The oligosaccharide blend also stimulated growth of the bacterium (FIG.2).

FIG. 1 illustrates the experimental results indicating a metabolicstimulation of a bifidobacterium (B. longum subsp infantis) in DMEMculture medium either without additional carbohydrates or withadditional glucose (Glc), or lacto-N-neoteraose (LNnT) or2′fucosyllactose (2FL) or 6′sialyllactose (6SL) or a blend of LNnT, 6SLand 2FL. (n=6; mean with SEM are shown; significance by ANOVA indicated(p<0.01). Note: Only the blend of LNnT, 6SL and 2FL stimulatedsignificantly the production of acetate.

FIG. 2 illustrates the experimental results showing in vitro growth of abifidobacterium (B. longum subsp infantis) in culture medium eitherwithout additional carbohydrates or with additional glucose (Glc), orlacto-N-neoteraose (LNnT) or 2′fucosyllactose (2FL) or 6′sialyllactose(6SL) or a blend of LNnT, 6SL and 2FL. (n=4; mean with SEM are shown;significance by ANOVA (p<0.02) indicated by small letters).

Study Model 2: Retrospective Epidemiologic Analysis of Breast MilkSamples and Occurrence of Allergic Diseases Up to the Age of 2 YearsApproach:

From a cohort of about 52 cesarean born infant mother pairs we analysedearly milk samples for amounts of specific oligosaccharides present. Tothis end defatted milk samples were diluted 10 to 100 times in water andanalysed by HPAEC (Dionex) equipped with a CarboPac PA1 column (Dionex)and an electrochemical detector. Oligosaccharide identification andquantification was done with authentic oligosaccharide standards. Weplotted absence or presence of (A) allergic diseases, (B) atopicallergies, (C) atopic eczema for infants against amounts of anoligosaccharide blend composed of 2FL, LNnT, and 6SL (FIG. 3) orcomposed of 2FL, LNnT, LNT (lacto-N-tetraose), 6SL and 3SL(3′sialyllactose) (FIG. 4).

FIG. 3 illustrates the experimental results showing in caesarean bornchildren absence (no) and occurrence (yes) of (A) allergic diseases, (B)atopic allergies, (C) atopic eczema up to 2 years of age. Plotted areamounts of a specific oligosaccharide blend measured in breast milk.Here the represented oligosaccharide blend is the sum of2′fucosyllactose (2FL), Lacto-N-neotetraose (LNnT) and 6′siaylllactose(6SL). (Statistical significance is indicated)

FIG. 4 illustrates the experimental results showing in caesarean bornchildren absence (no) and occurrence (yes) of (A) allergic diseases, (B)atopic allergies, (C) atopic eczema up to 2 years of age. Plotted areamounts of a specific oligosaccharide blend measured in breast milk.Here the represented oligosaccharide blend is the sum of2′fucosyllactose (2FL), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose(LNT), 6′sialyllactose (6SL) and 3′siaylllactose (3SL). (Statisticalsignificance is indicated)

Results:

We surprisingly found a trend to relatively low levels of specificoligosaccharide blends in early milk when children developed an allergicdisease (FIG. 3A, 4A). For atopic diseases and especially atopic eczemawe measured significantly lower levels of the oligosaccharide blends inearly milk of mothers whose children developed such allergies until theage of 2 years.

1. A method for the prevention and/or treatment of skin conditions andskin diseases comprising the step of administering a compositioncomprising at least one N-acetyl-lactosamine, at least one sialylatedoligosaccharide and at least one fucosylated oligosaccharide to anindividual in need of same.
 2. Method according to claim 1, wherein theN-acetyl-lactosamine is selected from the group consisting oflacto-N-tetraose and lacto-N-neotetraose.
 3. Method according to claim1, wherein the sialylated oligosaccharide is selected from the groupconsisting of 3′-sialyllactose and 6′-sialyllactose.
 4. Method accordingto claim 1, wherein the fucosylated oligosaccharide is selected from thegroup consisting of 2′-fucosyllactose, 3-fucosyllactose,difucosyllactose, lacto-N-fucopentaoses, lacto-N-difucohexaose I,fucosyllacto-N-hexaose, Difucosyllacto-N-hexaose I andDifucosyllacto-N-neohexaose II.
 5. Method according to claim 1, whereinthe composition comprises at least one probiotic bacterial strain. 6.Method according to claim 1, wherein the composition comprises at leastone prebiotic.
 7. Method according to claim 1, the composition isselected from the group consisting of a starter infant formula, aninfant formula, a follow-on formula, a baby food formula, an infantcereals formula and a growing-up milk.
 8. Method according to claim 1,wherein the individual is a healthy infants and healthy young children.9. Method according to claim 1, wherein the composition is administeredbefore and/or during a weaning period.
 10. Method according to claim 1,comprising the step of preventing and reducing the frequency and/oroccurrence and/or severity and/or duration of said skin diseases. 11.Method according to claim 1, comprising the step of promoting skinhealth and/or preventing skin dehydration and/or enhancing the hydrationof the skin and/or reducing skin rash, roughness and/or dryness and/orenhancing the oral tolerance to allergens.
 12. Method according to claim1 comprising the step of treating atopic dermatitis.
 13. Method for theenhancement of the oral tolerance to allergens in an individualcomprising the step of administering to the individual a compositioncomprising at least one N-acetyl lactosamine, at least one sialylatedoligosaccharide and at least one fucosylated oligosaccharide, as asynthetic nutritional agent.
 14. Method according to claim 12, whereinthe composition is a supplement.